Protein kinase A phosphorylates hepatocyte nuclear factor-6 and stimulatesglucose-6-phosphatase catalytic subunit gene transcription

Glucose-6-phosphatase is a multicomponent system that catalyzes the termina l step in gluconeogenesis. To examine the effect of the cAMP signal transdu ction pathway on expression of the gene encoding the mouse glucose-6-phosph atase catalytic subunit (G6Pase), the liver-derived HepG2 cell line was tra nsiently co-transfected with a series of G6Pase-chloramphenicol acetyltrans ferase fusion genes and an expression vector encoding the catalytic subunit of cAMP-dependent protein kinase A (PKA). PKA markedly stimulated G6Pase-c hloramphenicol acetyltransferase fusion gene expression, and mutational ana lysis of the G6Pase promoter revealed that multiple cis-acting elements wer e required for this response. One of these elements was mapped to the G6Pas e promoter region between -114 and -99, and this sequence was shown to bind hepatocyte nuclear factor (HNF)-6. This HNF-6 binding site was able to con fer a stimulatory effect of PKA on the expression of a heterologous fusion gene; a mutation that abolished HNF-6 binding also abolished the stimulator y effect of PKA, Further investigation revealed that PKA phosphorylated HNF -6 in vitro. Site-directed mutation of three consensus PKA phosphorylation sites in the HNF-6 carboxyl terminus markedly reduced this phosphorylation. These results suggest that the stimulatory effect of PKA on G6Pase fusion gene transcription in HepG2 cells may be mediated in part by the phosphoryl ation of HNF-6.